Mechanisms of Altered Calcium Signaling via ATP-Activated P2Y2 Receptor in Breast Cancer Cells

Abstract

The majority of breast cancer patient deaths occur when tumor cells migrate away from the primary tumors and metastasize. Tumor cells released from invasive tumor fronts can enter the bloodstream and become circulating tumor cells that disseminate to distant tissues during metastasis. At the invasive tumor front, cancer cells are under similar mechanical stimuli as non-tumorigenic epithelial cells at wound edges but show less coordinated migration. While most wound healing studies occur over hours and days, our previous studies have shown that ATP released from mechanically wounded cells initiates a calcium (Ca²⁺) signal within seconds that spreads to neighboring cells through activation of P2Y2 receptor. Our RNA sequencing data shows the P2Y2 receptor is downregulated in MCF10A mammary epithelial cells compared to MCF10A cells with mutations of PTEN knockout (-/-) and KRas activation. This P2Y2 downregulation was recapitulated in multiple aggressive breast cancer cell lines, MDA-MB-231 and MDAMB- 436, that is verified by immunoblotting. Fluorescence microscopy and plate reader analyses were used to measure Ca²⁺ concentrations after ATP-stimulation to mimic mechanical signals. Total fluorescence change was measured, and baseline corrected using Fluo-4, a fluorescent Ca2+ indicator. Phenotypic experiments examined the effect of rapid Ca2+ concentration changes on actin localization using phalloidin and 3-D cell dissemination from spheroids. Cytosolic Ca2+ increases significantly after ATP addition in MCF10A cells, while the cells treated with P2Y2 antagonist had a blunted response similar to mutant and metastatic cells. Non-tumorigenic cells stained with phalloidin showed actin localization and polymerization at cell edges after cytosolic Ca2+ increase. P2Y2 inhibitor blunted the actin response in MCF10A cells while metastatic and mutant cells had no major changes. We also demonstrate that P2Y2 inhibitor increases MCF10A spheroid dissemination in a 3-D matrix. Our data show that metastatic breast cancer cells have less P2Y2 expression, correlating with blunted ATP response and cytosolic Ca2+. The tumor microenvironment is known to have high concentrations of ATP, similar to a wound edge, and the lack of P2Y2 may allow cancer cells to avoid typical mechanical signals from the environment. Clarifying these molecular mechanisms of Ca2+ signaling and mechanotransduction could reveal new targets for cancer treatments.